FOXO3a/PI3K/Akt pathway participates in the ROS- induced apoptosis triggered by α-ZEL and β-ZEL

Zearalenone (ZEN), an estrogenic mycotoxin, is one of the most common food and feed contaminants. Also, its metabolites α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) are considered to induce oxidative stress, however its effect in prostate cells is not known yet. Our previous observations showed that forehead box transcription factor 3a (FOXO3a) expression is modified in hormone- sensitive cells in the response to mycotoxins, similar to the phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) pathway. Thus, this study evaluated the direct molecular effect of α-ZEL and β-ZEL in a dose of 30 µM in hormone-dependent human prostate cancer (PCa) cells with the focus of the involvement of FOXO3a and PI3K/Akt signaling pathway in that effect. We observed that both active metabolites of ZEN reduced cell viability, induced oxidative stress, cell cycle arrest and apoptosis in PCa cells. Furthermore, we observed that FOXO3a as well as PI3K/Akt signaling pathway participate in ZELs induced toxicity in PCa cells, indicating that this signaling pathway might be a regulator of mycotoxin-induced toxicity generally.

group of natural products that modulates the expression and action of FOXO3a in cells: apigenin 13 , resveratrol 14 , diosmetin 15 as well as sulforaphane 16 .Also mycotoxins have been reported to modulate the FOXO3a in cells.DON was reported to induce apoptosis in small-intestine cells in pigs via modulation of FOXO3a signalling pathway and ERK1/2 17 .The regulation of apoptosis process as well as mitochondrial toxicity and cell death by FOXO3a and ROS/JNK pathway in DON action was also reported in another study 18 .FOXO3a was also reported to participate in fusaric acid and fumonisin B1-induced apoptosis in human hepatic cells HepG2 19 .Nevertheless, the interplay between FOXO3a and PI3K/Akt signalling pathway in the action of mycotoxins in PCa cells has not been elucidated yet.Thus, we decided to evaluate the interplay of FOXO3a and PI3K/Akt in PCa cell in the response to the two most common and potent ZEN metabolites: α-ZEL and β-ZEL.For this purpose, we generated FOXO3a-PC3 cells in which the PI3K/Akt signalling pathway was blocked with selective chemical inhibitor LY294002.The toxic effect of ZELs was evaluated with cell viability, oxidative stress, apoptosis and cell cycle evaluation.

α-ZEL and β-ZEL induce toxicity and upregulate expression of FOXO3a and Akt in PC3 cells
Firstly, we assessed if α-ZEL and β-ZEL induce cytotoxicity in PC3 cells.After 24 h we observed that both mycotoxins caused a significant decrease in cell viability (Fig. 1a).As expected β-ZEL showed less toxic effect than α-ZEL, in the same concentration range.For the tested dose range IC 50 value was not reached for β-ZEL, whereas for α-ZEL it accounted 45.19 µM.
Next, we evaluated if tested mycotoxins might affect the expression of FOXO3a and Akt in PC3 cells.For this purpose we chosen a range of concentrations: 30 µM, 10 µM and 1 µM of α/β-ZEL and treated the cells for 24 h.The results showed (Fig. 1b-d) that both α-ZEL and β-ZEL increase the expression of FOXO3a and simultaneously decreased the expression of phospho-Akt.We observed significant increase in the expression of FOXO3a after treatment with 30 µM α-ZEL as compared to control (**p < 0.01) as well as other tested doses of α-ZEL (*p < 0.05).A similar effect was caused by 30 µM of β-ZEL.A not significant decrease in the expression of phospho-Akt was observed for the highest tested dose of mycotoxins, whereas for lower tested doses a slight increase was observed.

FOXO3a/PI3K/Akt participates in α-ZEL and β-ZEL ROS-induced apoptosis in PCa cells
To confirm the role of FOXO3a in the mycotoxin-induced toxicity, knockdown of FOXO3a was generated with CRISP-Cas9 method.The sufficiency of silencing was confirmed on Western blot and was sufficient for further experiments (Fig. 2a).Next, the cytotoxicity of α-ZEL and β-ZEL was assessed in generated cells.First of all, any significant change in cell viability between PC3-FOXO3a-and PC3-CNT was observed (Fig. 2b).A statistically significant decrease in cells response to α-ZEL was observed in both cell lines as compared to non-treated cells for concentration range 100-30 µM (*p < 0.05, ***p < 0.001) for PC3-FOXO3a-and 100-50 µM (***p < 0.001) for PC3-CNT.The observed statistically significant decrease in cell viability was in the range of 12% to 72% for α-ZEL and 26% to 40% for β-ZEL in PC3-FOXO3a-cells.The statistically significant decrease in cell viability in PC3-CNT was in range of 30% to 67% for α-ZEL.In case of β-ZEL a similar trend was observed, but was significant only for 70 µM and 100 µM (***p < 0.001) for PC3-FOXO3a-.Based on these results the dose of 30 µM of α-ZEL and β-ZEL were chosen for the rest of experiment.
To further understand the role of FOXO3a in ZELs toxicity, the role of FOXO3a and PI3K/Akt was elucidated in oxidative stress, apoptosis and cell cycle regulation.For these experiments the dose of 30 µL of mycotoxin was chosen.The rationale for such a relative high dose of mycotoxins metabolites was based on cell-viability assay, and the fact that aim of this study is to elucidate the molecular mechanism of action of mycotoxins.Both generated cell lines PC3-FOXO3a-and PC3-CNT, were then treated with mycotoxins and/or selective PI3K/ Akt inhibitor LY294002.
Firstly, we evaluated how the silencing of FOXO3a modulated the response of cells to ZELs (Fig. 3a,b).We observed that PC3-CNT cells responded similarly to PC3 cells, where treatment with ZELs resulted in the increased expression of FOXO3a.In all cases addition of LY294002 resulted in the increased expression of FOXO3a as compared to mycotoxin treated-PC3-CNT cells.A similar effect was observed in PC3-CNT cells not treated with mycotoxin.In case of PC3-FOXO3a − cells no changes in the expression of FOXO3a were present, also no changes after addition of LY294002 were observed indicating that FOXO3a might serve as a crucial agent in PI3K/Akt activation.
Next, we move forward and evaluated the cell viability (Fig. 3c).In that case silencing of FOXO3a in cells resulted in no changes in the cell viability as compared to PC3-CNT cells in case of both tested mycotoxins.In all cases, a significant decrease in cells viability was observed after addition of LY294002, also for control cells, confirming the fact, that PI3K/Akt signaling pathway is necessary for cell growth.
Further we conducted ROS (Fig. 3d,e) and apoptosis analysis.We observed that lack of FOXO3a resulted in a slightly decreased number of ROS positive cells, however not significant for ZELs treatment, but significant for control cells (*p < 0.05).Addition of LY294002 resulted in a significant changes between PC3-FOXO3a-and PC3-CNT cells.First of all, a lower extend of ROS positive cells was observed.This fact was possibly caused by the fact that the cells were not dividing, as observed in light microscopy (data not shown).Then, treatment with ZELs and LY294002 resulted in a significant decrease in the number of ROS positive cells in PC3-FOXO3a-as compared to PC3-CNT cells (***p < 0.001 and **p < 0.01 for α-ZEL and β-ZEL, respectively).This effect was contradictory for the one observed for not treated cells (***p < 0.001).To elucidate the oxidative stress induced   S1.Changes in viability after treatment with mycotoxins and/or selective PI3K/Akt inhibitor LY294002 was evaluated with toxicity assay (MTT assay) after 24 h incubation in PCa cells (c).The changes in ROS production in PCa cells was expressed as the percentage of gated cells (d).The representative histogram of cells distribution according to ROS (e).The relative expression of SOD1, SOD2 and PARP1 after ZELs and/or LY294002 treatment (f).The relative expression of SOD1, SOD2 and PARP1 evaluated with Western blot (g).The values are expressed as the mean ± SE; One-way ANOVA with Bonferroni correction was used for statistical analysis, p < 0.05 was considered statistically significant, *** p < 0.001, **p < 0.001, *p < 0.05 as compared to the control.α-ZEL-α-zearalenol, β-ZEL-β-zearalenol, SOD1-superoxide dismutase 1, SOD2-superoxide dismutase 2, PARP1-poly(ADP-ribose) polymerase-1, GAPDH-glyceraldehyde 3-phosphate dehydrogenase, Cnt-control (not treated cells).1, 2-30 µM α-ZEL; 3, 4-30 µM α-ZEL + LY294002; www.nature.com/scientificreports/by ZELs we evaluated the expression of oxidative response enzymes and observed that different superoxide dismutases were activated by α-ZEL and β-ZEL (Fig. 3f,g).β-ZEL significantly induced SOD1 expression (~ 1.95 fold, ***p < 0.001), whereas α-ZEL caused a significant increase in SOD2 expression (~ 1.68 fold, ***p < 0.001) as compared to non-treated cells in both cell lines.In all cases PC3-FOXO3a-cells were more sensitive to ZELs, whereas addition of LY294002 resulted in a decreased expression.Similar changes were observed on protein level, however not significant.In case of PARP1 expression a significant decrease was observed for both ZELs, as well as higher in PC3-FOXO3a-cells as compared to PC3-CNT cells.In all cases addition of LY294002 resulted in a significant decrease in the expression (Table 1.).Then, we decided to evaluate if the increase in the ROS production in cells might be associated with apoptosis in cells.So, firstly we stained the apoptotic cells and counted them with flow cytometry (Fig. 4a,c).We observed that both ZELs significantly increased the number of apoptotic cells (*p < 0.05), and as expected α-ZEL was more toxic than β-ZEL in the same concentration of 30 µM.In all tested conditions PC3-FOXO3a-cells were not significantly less susceptible to apoptosis-inducing condition as compared to PC3-CNT cells, visible also in case of control cells.Blocking of PI3K/Akt resulted in a significant decrease in the number of apoptotic cells in case of both mycotoxins treatment (**p < 0.01, ***p < 0.001) as well as control (**p < 0.01) as compared to cells not treated with LY294002.The observed decrease in the number of apoptotic cells was proportional to the ZELs induced effect.
The observed induction of apoptosis might be associated with changes in the mitochondrial potential, so we decided to elucidate the changes in the mitochondrial potential of cells (Fig. 4b,d).Firstly, we observed that α-ZEL did not significantly increase the number of depolarized cells.Interestingly, a higher increase was observed for β-ZEL, especially in PC3-CNT cells (***p < 0.001).A significant difference was observed between PC3-FOXO3a-and PC3-CNT cells treated with 30 µM β-ZEL (***p < 0.001).When LY294002 was added, a significant decrease in the number of depolarized cells was observed as compared to the same cells treated without LY294002 (***p < 0.001).A contradictory effect was observed for PC3-FOXO3a-cells treated with α-ZEL and control cells PC3-CNT for which addition of LY294002 resulted in the increased number of apoptotic cells.
Next, we evaluated the expression of Casp3 and Casp7 (Fig. 4e).Similarly, a different expression regulation was observed after treatment with α-ZEL and β-ZEL.α-ZEL did not change the expression of Casp7 whereas the expression of Casp3 was not significantly decreased.Any significant changes were observed between PC3-FOXO3a-and PC3-CNT cells.An insignificant increase in the expression of Casp7 was observed after treatment of both cell lines with 30 µM of β-ZEL.In case of Casp3 the expression was almost no changed as compared to control.Addition of LY294002 decreased the expression of both Casp3 and Casp7 in a significant manner.In case of mycotoxins treatment the decrease in the expression of Casp3 was significant in PC3-FOXO3a-cells after β-ZEL treatment as compared to PC3-CNT cells (**p < 0.01).A similar effect was observed for Casp7 expression both for β-ZEL (***p < 0.001) as well as α-ZEL treatment (*p < 0.05).No such effect was observed for control cells treated with LY294002.
Later, the progression of cell cycle was evaluated (Fig. 5a,b).In case of control cell line a significant decrease in the number of cells in S phase was observed (***p < 0.001).Treatment of α-ZEL and β-ZEL caused a different modulation of cell cycle progression.α-ZEL caused a significant increase in the number of cells in G2/M cell cycle phase as compared to respective control (***p < 0.001) with simultaneous significant decrease in the number of cells in G0/G1 and S cell cycle phase (***p < 0.001).In all treatments with α-ZEL there was a significant decrease in the number of cells in PC3-FOXO3a-cells as compared to PC3-CNT cells in G2/M cell cycle phase with increase in G0/G1 cell cycle phase.Contradictory, β-ZEL caused a significant decrease in the number of cells in S and G2/M cell cycle phase (***p < 0.001) and increased the number of cells in G0/G1 cell cycle phase (***p < 0.001).Addition of LY294002 in case of control cells significantly modulated the cell cycle progression: decreased the number of cells in S and G2/M cell cycle phase in favor of the increase of the number of cells in G0/G1 cell cycle phase (***p < 0.001).A similar effect was observed for α-ZEL, whereas for β-ZEL such change was not observed.
The modulation of cell cycle progression was also visible in the modulation of cell cycle genes: CDKN1A, CDC2 and CCNB1 (Fig. 5c).In case of CDKN1A a not significant increase in the expression was observed after α-ZEL treatment, with no changes between cell lines as well as LY294002 addition.After treatment with β-ZEL a significant decrease in the expression was observed as compared to control cell lines (0.52 fold, ***p < 0.001) Table .1.The integrated optical density of the expression of proteins associated with oxidative stress.The results are expressed as a mean.FOXO3a-forkhead box O3, GAPDH-anti-glyceraldehyde 3-phosphate dehydrogenase, SOD1-superoxide dismutase 1, SOD2-superoxide dismutase 2, PARP1-poly (ADP-ribose) polymerase 1, α-ZEL-α-zearalenol, β-ZEL-β-zearalenol, Cnt-control, non-treated cells.and not significant difference between PC3-FOXO3a-and PC3-CNT cells.In case of control cells treatment with LY294002 resulted in a significant decrease in the expression, whereas for α-ZEL no change was visible and in case of β-ZEL a not significant increase was observed.In case of CDC2 expression ZELs decreased its expression significantly as compared to control (~ 0.26 fold, ***p < 0.001) and similarly to CDKN1A expression, LY294002 decreased the expression, whereas in case of ZELs increased the expression.The difference between PC3-FOXO3a-and PC3-CNT cells was also visible but not significant.A similar expression pattern was observed for another cell cycle modulator CCNB1.All the tested treatments resulted in a significant decrease of the expression of CCNB1 as compared to control respective cell line.The highest decrease in the expression was observed for control treatment with LY294002 (~ 0.4 fold, ***p < 0.001) whereas for ZELs any differences were not observed.Subsequently, we decided to evaluate the expression of SIRT1 and Akt as a main regulators of FOXO3a in cells (Fig. 6a,b).We observed that α-ZEL increased the expression of SIRT1 as compared to respective control, whereas addition of LY294002 resulted in a higher increase, however not significant.In case of β-ZEL a contradictory effect was observed with a visible difference between PC3-FOXO3a-and PC3-CNT cells.Itself 30 µM of β-ZEL caused almost no change in the expression of SIRT1 in PC3-FOXO3a-cells, whereas in PC3-CNT cells the increase was observed.Similarly to control cells, addition of LY294002 resulted in a slight, not significant decrease, however a pattern of change of the SIRT1 expression was sustained.The expression of p-Akt/Akt was evaluated on Western blot and showed that in PC3-CNT cells α-ZEL slightly increased the expression of p-Akt, whereas β-ZEL almost no changed it.As suspected, lack of FOXO3a in cells resulted in a decrease in the expression or all tested mycotoxins as well as control condition, whereas LY29002 decreased it even more.

Discussion
FOXO3a is considered a potential target for anticancer therapy.This is due to its well-known involvement in the regulation of cell proliferation, transformation, cell apoptosis and autophagy.Its interaction with other major cell signaling pathways also plays an important role: PI3K/Akt, epidermal growth factor receptor (EGFR), mitogenactivated kinase (MAPK) or ERK kinase.Multiple nutrients have previously been reported to modulate FOXO3a in PCa 9 .Most of them interacts with PI3K/Akt signaling and modulates the cell cycle progression, proliferation and induces apoptosis in cells 20 .As showed previous studies that also mycotoxins including DON and ZEN might affect the expression of FOXO3a in PCa cells 17,21 .However none of that studies considerated the active metabolites of ZEN.The present study provides an information of the involvement of both FOXO3a as well as PI3K/Akt signaling pathway in ZELs-induced toxicity in PCa cells.It was also observed that both mycotoxins have different molecular mechanisms induced by ROS in PCa cells, related to the FOXO3a and PI3K/Akt signaling pathway.
The hydroxyl derivatives of ZEN: α-ZEL and β-ZEL are known from estrogenic potency in cells, even higher than ZEN itself in case of α-ZEL 22 , ability to induce oxidative stress 23 and DNA damage 24 .The justification for undertaking research assessing the impact of ZEN metabolites on cells is the fact that they are detected in human and animal samples 25 .Moreover, the transformation of ZEN to ZELs might happen in liver, lung, kidney as well as prostate 25 .Due to the fact that PTEN loss corresponds with hormone insensitive PCa cells, in this study we used androgen-independent cell line: PC3.Firstly, we confirmed our assumption that ZELs increases the expression of FOXO3a in a significant manner in PCa cells.A similar, dose-dependent effect has been observed previously by Kang et al. 17 in case of DON in a dose of 2 and 1 µg/mL in porcine IPEC-J2 cells.Sang et al. reported that ZEN in a dose of 20 µM increased the acetyl form of FOXO3a, which serves as the active one in human embryo kidney HEK293 cells 21 .It also seems that the role of FOXO3a might be different in normal prostate cells in which ZEN decreased the expression of FOXO3a in a dose-dependent manner 26 .This observation on the other hand was not evaluated for ZEN metabolites as well as in this study we considered the protein level of FOXO3a, not the gene level, which also might be different.www.nature.com/scientificreports/As we have shown, most of the toxic effect of ZEN is associated with induction of oxidative stress, apoptosis and cell cycle modulation in cells 27 .In our present study we observed that α-ZEL is more toxic to β-ZEL as well the mechanism of apoptosis as well as cell cycle arrest is different in PCa cells.First of all we observed that both mycotoxins induced oxidative stress in cells manifested by the increased number of ROS-positive cells.This fact is associated with the previous observation that ZEN itself induces oxidative stress in PCa cells observed in prostate 26 and liver cells 28 .The observed increase in the number of ROS-positive cells after ZELs treatment was associated with modulation of the expression of SOD1/SOD2 and PARP1 in PC3 cells.ZEN was reported to modulate the expression of SOD1 in other PCa cells: LNCaP as well DU-145 cells 27 .The decreased expression of SOD1 was observed in porcine granulosa cells after treatment with ZEN 29 as well as weanling piglets 30 .The induction of oxidative stress was also observed in HEK293 cells by both ZELs, however in a significantly higher concentration than observed by us: 150 µM and 240 µM of α-ZEL and β-ZEL, respectively 31 .We also observed the decrease in PARP1 expression suggesting that generation of oxidative stress might be also associated with DNA damage, which was observed previously for other mycotoxin DON 32 .However it was noted that the same dose of α-ZEL or β-ZEL caused a different expression pattern.This fact might be explained by a different susceptibility of cells to mycotoxin-induced toxicity.For both mycotoxins we observed induction of apoptosis in cells, however to higher extend by α-ZEL for which almost no changes were observed in mitochondrial potential as well as caspase 3 and 7 expression.In RAW264.7 macrophages the ROS-induced apoptosis by ZELs was not associated with caspase activation and similarly to our results β-ZEL more significantly induced the mitochondrial depolarization, even when being less toxic to the cells 4 .A different mechanism of cellular death caused by ZELs was also visible in cell cycle analysis.The modulation of cell cycle by ZEN is associated with cell cycle arrest in G2/M cell cycle phase 33 .We also observed a significant arrest in G2/M cell cycle phase in cells treated with α-ZEL, however in β-ZEL treated cells the cell distribution was different indicating cell cycle arrest in G0/ G1 cell cycle phase.This observation is consistent with previous one made by Tiemann et al. who observed that 30 µM of β-ZEL increased the number of cells in G0/G1 phase 22 in porcine endometrial cells.Similar observation for both ZELs was made by Minervini et al. who observed that both ZELs induced an increase in the number of cells in sub-G0 cell cycle phase in granulosa cells 34 .
The modulation of PI3K/Akt signalling pathway by mycotoxins was reported before 7 .It was previously showed that NRF2 activation due to exposure to ZEN is mediated by PI3K/Akt 7 .In macrophages p53, JNK and p38 kinases were activated upon exposure to ZELs 4 , although the authors did not consider involvement of PI3K/Akt pathway in that effect, MAPK kinases has been known to be downstream target of PI3K/Akt signalling cascade 35 .Both ZEN and DON were reported to induce the autophagy and apoptosis of cells via modulation of PI3K/ Akt 36 .The involvement of SIRT1 reported to protect cardiac cells against ZELs-induced apoptosis 37 seems to also supports our hypothesis.The authors suggested that activation of SIRT1 protects the cells against the cytotoxic effect of mycotoxins.In our study we observed that in case of β-ZEL silencing of FOXO3a significantly decreased expression of SIRT1 in the response to the mycotoxin.Previous studies suggested that observed toxicity of ZEN might be associated with epigenetic changes: DNA methylation and histone methylation 38 .SIRT1 is a class III histone deacetylease which regulates tissue homeostasis and deacetylation of histone and not-histone targets in many diseases 39 .The observed modification of the expression of SIRT1 suggests that ZELs, similarly to ZEN might affect epigenetic modification in cells, as a part of its toxic mechanism.Also we observed that FOXO3a itself only in cell cycle significantly reduced the toxic effect of ZELs in cells.Whereas a simultaneous blockage of FOXO3a as well as PI3K/Akt resulted in a significant decrease in the proliferation of cells and reduction of oxidative stress as well as induction of apoptosis.This results seems to be in line with the work of Das et al. who showed that inhibition of Akt promotes FOXO3a-dependent apoptosis of PCa cells 40 .Based on the fact that also other mycotoxins like DON were reported to induce apoptosis via FOXO3a signalling pathway in different cell line, our results indicate that FOXO3a-PI3K/Akt signalling pathway might be involved in the general cytotoxic effect of mycotoxins, observed over last few years extensively in different cell lines.

Conclusions
This is the first study which showed that both active metabolites of ZEN induces toxicity in PCa cells and FOXO3a/PI3K/Akt signalling participates in that effect.The results showed that both metabolites possesses a different cytotoxic potential in cells and the observed apoptosis in cells seems to be regulated by a different mechanism, however this statement needs further studies to be confirmed.

Silencing of FOXO3a
Human FOXO3 CRISPR/Cas9 KO plasmids, and control (Scramble) (Scr) were purchased from Applied Biological Materials Inc. (BC, Canada).Three different FOXO3 CRISPR/Cas9 KO plasmids were tested, each encoding the Cas9 nuclease and a target-specific 20 nt guide RNA (gRNA) designed for maximum knockout efficiency: target 1: 5′-CCC GCT CTC TCC GCT CGA AG-3′, target 2 5′-TTT GTC CGG GGA GCT CTC GA-3′ and target 3 5′-CAG AGT GAG CCG T TTG TCC G-3′.CRISPR/Cas9 KO Plasmids and Control CRISPR/Cas9 Plasmid were transfected into PC3 cells using ViralEntry™ Transduction Enhancer (Applied Biological Materials Inc., Canada) according to manufactures' protocol.Briefly, PC3 cells were seeded onto 12-well plate.After 24 h and reaching 60-70% confluency, the culture media were exchanged to experimental medium with plasmid and ViralEntry™ Transduction Enhancer.At 48 h post-transfection, cells from each well were transferred to the next two wells.The next day medium was exchange to 1 mL of selection medium (complete medium containing 2 μg/mL of puromycin).Successful transfection of the FOXO3 and Control plasmids was verified with Western blots.Target 1 was the most efficient and was selected to further experiments.
PC3-FOXO3a-and PC3-CNT cells apoptosis was determined using the Muse™ Annexin V and Dead Cell Kit (Merck Millipore, Burlington, MA, USA).The cells (6 × 10 4 /well) were seeded on 12-well plates and left to reach 90% confluence.After exposure to 30 µM α-ZEL, 30 µM α-ZEL + LY, 30 µM β-ZEL, 30 µM β-ZEL + LY, LY or medium alone the cells were detached and suspended in 100 μL of culture medium.The assay was performed according to the manufacturer's instructions.The analysis was performed in three independent experiments.
Muse™ MitoPotential Assay (Merck Millipore, Burlington, MA, USA) which evaluates the polarization of mitochondria was used to measure the changes in mitochondrial membrane potential (ΔΨm) in PC3-FOXO3aand PC3-CNT.Simultaneous use of 7-AAD allows evaluation of cell membrane integrity.Cells (approximately 6 × 10 4 /well) were seeded on 12-well plates and left to reach 90% confluence.Then, the cells were treated with 30 µM α-ZEL, 30 µM α-ZEL + LY, 30 µM β-ZEL, 30 µM β-ZEL + LY, LY or medium alone for 24 h.The assay was performed as recommended by the manufacturer.The probes were standardized against control probes.The experiment was repeated three times.
Propidium iodide (PI) staining in the presence of RNAse was used to evaluate the percentage of cells in the G0/G1, S and G2 phase of cell cycle with the Muse® Cell Cycle Assay Kit (Merck Millipore, Burlington, MA, USA). 3 × 10 5 /well cells (PC3-FOXO3a-and PC3-CNT) were seeded on 6-well plates and cultured to reach 90% confluence.Next, treated with 30 µM α-ZEL, 30 µM α-ZEL + LY, 30 µM β-ZEL, 30 µM β-ZEL + LY, LY or medium alone for 24 h and trypsinized.The Cell Cycle Assay was conducted according to manufacturer's recommendations.Cells were analyzed on Muse™ Cell Analyzer (Merck Millipore, Burlington, MA, USA) and compared to control cells.The results were expressed as the percentage of cells in each cell cycle phase.The experiment was conducted in triplicate.

Statistical analysis
Results are expressed as mean ± SE.The one-way ANOVA test was used to calculate statistically significant differences.p < 0.05 was considered as statistically significant.GraphPad Prism software (GraphPad Software, La Jolla, CA, USA) was used to carry out all statistical analyses.

Figure 4 .Figure 5 .
Figure 4.The influence of α-ZEL and β-ZEL on the number of apoptotic cells and mitochondrial potential in PC3-FOXO3a-and PC3-CNT cells.The changes in the number of apoptotic cells (a) and mitochondrial potential (b) after ZELs and/or LY294002 treatment were measured and expressed as the percentage of gated cells; The representative histogram of apoptosis profile (c) and mitochondrial potential (d).The relative expression of Casp3 and Casp7 after ZELs and/or LY294002 treatment.The values are expressed as the mean ± SE; One-way ANOVA was used for statistical analysis.p < 0.05 was considered statistically significant, *** p < 0.001, **p < 0.001, *p < 0.05 as compared to the control.α-ZEL-α-zearalenol, β-ZEL-β-zearalenol, Casp3caspase 3, Casp7-caspase 7, Cnt-control (not treated cells).